DNaseI have affect with RT-PCR or not - (Mar/17/2005 )
I have been using Qiagen's RNeasy mini kit for RNA isolation. Next, sample is treated a DNaseI and purified by using phenol/choloform method. I try to RT-PCR and PCR of the sample but I can not see band. This indicated that my mRNA degrade by phenol/choloform or not. If purification method of sample are not used before RT-PCR, what happen. Thanks so much
I can see total RNA, before treat sample with DNase I, from denature gel electrophoresis
I have few question to ask:
1) since you are using RNA extraction kit, why you were doing PCR? Do you means you run RT-PCR first, don see band and followed by second PCR?
2) Have you get this set of primer work before?
DId you use RNase inhibitor. And how are the RNAs are stored? Keep in mind that may still present in you RNA prep. Use of RNase inhibitor and RNA storage buffers, and put them in -80C.
Thank you for every one answer me
After RNA extraction by those kit, I try to normal PCR which can see band. Thus, samples were treated with DNaseI and purify by phenol choloform. Next, I try to normal PCR again that can not to see band and then RT-PCR is made. I can not to see band from RT-PCR also. So, I thinks that mRNA is degraded by phenol/choloform method or not. Purification method is not used before RT-PCR, I will success to amplify or not.
Primer is not disized to specific primer but can amplify a gene well from genomic DNA
For inhibitor, I thinks that RNA extraction kit already contained RNase inhibitor. Instument for use in RNA preparation were also double autoclave (tip and microtube) and treated with DEPC (reagent solution).
best my regards